ZAP-70 is a cytoplasmic protein tyrosine kinase that plays a critical function in T cell antigen receptor (TCR) signaling and in most aspects of T cell biology. Considerable effort has been expended by the big pharma and biotech industries to develop ZAP-70 catalytic inhibitors, without success. Thus, it has also been difficult to validate ZAP-70 as a therapeutic target. We have recently developed a model chemical genetic inhibitor system for ZAP-70 in cell lines and in mice in which a bulky inhibitor is used to specifically target a mutant allele of ZAP-70. Work to date supports the notion that ZAP-70 function is critically important in naove and memory T cell function, but interestingly not in T regulatory cells. However, we have not yet extended our studies to preclinical disease models and further validation is needed. In addition, a new approach towards developing a ZAP-70 inhibitor is needed since the wild-type kinase catalytic site does not seem amenable to inhibitor development. Following TCR stimulation, the tandem SH2 domains of ZAP-70 associate with dual tyrosine phosphorylated residues located within conserved sequence motifs (referred to as ITAMs) of the cytoplasmic domains of the CD3 and zeta chains. Once associated with the ITAMS, ZAP-70 is activated by its phosphorylation by the Src kinase Lck and by its own trans- autophosphorylation. Recent mutagenesis and structural studies from our labs on the full-length ZAP-70 molecule suggest that, prior to association with the ITAMs, ZAP-70 is in an autoinhibited conformation that results from several intramolecular interactions. Moreover, our new insights suggest a conformational change in ZAP-70 is likely to occur during its binding to the TCR. Based on these recent studies, we now have the opportunity for a new approach towards developing a novel and potentially more specific allosteric inhibitor of ZAP-70 function. Thus, we propose to: 1) identify a lead compound that is a specific allosteric inhibitor of ZAP-70 and compare its efficacy on blocking signaling pathways by the model catalytic site inhibitor that we have developed;and, 2) using the analog-sensitive mutant of ZAP-70 in mice that we have established, we will determine the preclinical utility of ZAP-70 inhibition in arthritis, systemic lupus erythematosus and experimental autoimmune encephalomyelitis.